(A) Field of the Invention
The present invention concerns an immobilized enzyme, the production thereof and its use.
(B) State of the Art
Nowadays several formulations of immobilized enzymes are commercially applied in production processes, one of the possibilities is an immobilization method for enzymes using e.g. gelatin and glutaric dialdehyde, see U.S. Pat. No. 3,838,007. Gelatin as gelling agent is often chosen because it is relatively cheap compared to other gelling agents and is abundantly available. Cross-linking with e.g. glutaric dialdehyde is carried out to obtain sufficiently rigid particles. In this way it was possible to immobilize non-proteolytic enzymes.
The immobilization process comprises the following steps:
a crude enzyme containing biomass of about 4% (w/v) is mixed with a gelling agent and heated to slightly above the melting temperature of the gelling agent, for gelatin slightly above 40.degree. C. The final concentration of the gelling agent is about 8% (w/v); PA1 the gelling agent-water-enzyme and/or microbial cells containing mixture is subsequently prilled into a cold, water-immiscible solvent e.g. butyl acetate; PA1 after collecting the coagulated spherical enzyme-containing particles several washings with a water-miscible organic solvent e.g. acetone or ethanol may be carried out in order to dehydrate partially the particles. The enzyme particles so formed keep their integrity at temperatures below 12-15.degree. C. PA1 after these washings the excess of the organic solvent is removed by filtration, gravity sedimentation, centrifugation or decantation and the enzyme particles are resuspended in a mixture of cold water and acetone or ethanol and cross-linked with a bifunctional or poly-functional protein reagent such as glutaric dialdehyde. For example glutaric dialdehyde in a concentration of 0.5-5% (v/v) is sufficient; PA1 finally, the excess of the cross-linking agent and other soluble impurities are washed out with water and the immobilized enzyme particles are either dried after dewatering with e.g. ethanol or dried directly in a fluid bed or tumbling drier or preferably transferred to a propylene glycol-water mixture and drained such that at least 25% (v/v) concentration has been reached in the particle. PA1 (a) fermenting a strain of microorganism to produce a broth containing the enzyme; PA1 (b) adding a gelling agent to the broth formed in step (a); PA1 (c) adding 1 to 5% (w/v) active carbon; PA1 (d) forming an enzyme-gelling agent preparation in particulate form; and PA1 (e) cross-linking the gelling agent. PA1 less than 3% (w/v) gelatin, the solution does not coagulate during prilling; PA1 more than 10% (w/v) gelatin, the solution is too viscous. PA1 1% (w/v) of active carbon already gives an improvement in the strength of the formed particle; PA1 more than 5% (w/v) of active carbon: the solution becomes too viscous.
Another method of immobilizing enzymes, cells and/or a combination thereof is described in U.S. Pat. No. 4,163,691. A mixture of a slurry of microbial cells containing glucose isomerase, an endocellular enzyme, at a temperature less than about 55.degree. C. and 3 to 20 wt % of gelatin as a gelling agent passes a die to form threads into cold water which gels the threads. Instead of water, mixtures with organic solvents such as ethanol, methanol, acetone and water immiscible organic solvents such as ethylacetate,butylacetate and petroleum ether may be used which leads in many cases to reduced activity losses. The threads have a diameter of about 0.4 to 2 mm.
Subsequently the threads are cross-linked with 0.5 to 5% glutaric dialdehyde based on starting mass of the slurry and then cutting said cross-linked threads into pieces having a length of 0.4 to 10 mm.
An example is the preparation of an immobilized glucose isomerase on commercial scale. Also other enzymes with or without the presence of microbial cells may be immobilized with the use of this technique. As source of the enzyme glucose isomerase a strain of Actinoplanes missouriensis can be applied as described in U.S. Pat. 3,834,988. Submerged fermentation is carried out at neutral pH under aerobic conditions.
The immobilized glucose isomerase is applied in a plug flow or packed bed reactor at a temperature of 40 to 5.degree. C. In this reactor glucose is converted into fructose during down flow passage over a column packed with immobilized enzymes. Packed beds of about 5 m high are often applied. The mechanical strength (compressibility) of the immobilized enzyme particles determines the pressure drop allowed over the column. When glucose isomerase immobilized in gelatin is used, the particles have a size of 1.4 mm or more for the prilling process and a diameter of 1.4 mm or more and a length of 2-10 mm for the extruded particles. Smaller particles would be deformed under influence of the pressure drop over the column and subsequently would plug the column whereas the use of larger particles would lead to low conversions because the rate limiting step of the conversion reaction is determined by the diffusion in the particles.
The above described system dates back to the early and mid 1970's and up to now no improvements to this system have been made. In order to obtain higher conversion rates, the particles should be smaller and therefore more rigid. It is the aim of the present invention to provide more rigid particles.
In order to obtain more rigid particles the following compounds were added to the immobilized enzyme particles to determine whether they increase the rigidity of the particles: aluminium oxides, silicates, sulphur, filter aids, casein, yeast cells, starch, agar, lignocellulose, alginate and pectins. However, none of these additives were found to be effective. Often prilling or extruding of enzyme/gelling agent/additive was less effective due to high viscosities or surface tension changes. In some cases even weaker particles were formed.